The overall goal of this research is to learn the role that thionucleotides play in transfer RNA function. This will be accomplished through the use of the C6 mutant of Escherichia coli, which produces sulfur-deficient tRNA when starved for cysteine. Using this tRNA we obtained the first evidence of a potential role for thiobases in aminoacylation. Sulfur-deficient tRNA exhibits a 3-fold increase in the formation of isoleucyl-tRNA, and preliminary experiments suggest that this is due to a single tRNA ile species. The tRNA ile isoacceptors will be purified from fed and starved cells and their structural properties, modified nucleoside content and sequences compared. Using the pure tRNA ile species we will try to determine which step in aminoacylation is facilitated, namely, tRNA binding, ile-tRNA formation, or product release. Physical and chemical methods will be used to determine if a conformational change underlies the increased catalytic behavior of sulfur-deficient tRNA ile. Further, the interaction of this tRNA in other reactions of protein synthesis will be investigated, such as: interaction with transfer factors, ribosome binding, codon-directed binding, codon specificity, and finally, the ability to translate mRNA in cell-free systems. The activity of the enzymes of the ilv operon will be determined during cysteine starvation to see if the regulation of protein synthesis is altered under conditions in which the fast tRNA ile accumulate. Sulfur will be replaced in deficient tRNA using sulfurtransferases, to see if this reverses any of the functional alterations in aminoacylation, protein synthesis, etc. Other altered tRNA species in sulfur-deficient tRNA will be detected using RPC-5 column chromatography, and then isolated by a previously reported procedure. The functions of unique tRNA species will be determined in order to enlarge our understanding of the role that modified nucleotides play in this important molecule.